The present invention relates generally to methods and compositions for the detection of cervical cancer. More specifically, the present invention relates to cervical cancer-associated proteins which act as cellular markers useful (i) in detecting cervical cancer, and (ii) as molecular targets for cervical cancer therapy.
Cancer of the uterine cervix is one of the most common malignancies in women and remains a significant public health problem throughout the world. In the united States alone, invasive cervical cancer accounts for approximately 19% of all gynecological cancers (Miller et al. (1993) in xe2x80x9cSurveillance Epidemiology, and End Results Program cancer Statistics Review: 1973-1990xe2x80x9d, NIH Pub. No. 93-2789, Bethesda, Md.: National Cancer Institute). In 1996, it is estimated that there will be 14,700 newly diagnosed cases and 4900 deaths attributed to this disease (American Cancer Society, Cancer Facts and Figures 1996, Atlanta, Ga.: American Cancer Society, 1996). In many developing countries, where mass screening programs are not widely available, the clinical problem is more serious. Worldwide, the number of new cases is estimated to be 471,000 with a 4 year survival rate of 40% (Munoz et al. (1989) xe2x80x9cEpidemiology of Cervical Cancerxe2x80x9d in xe2x80x9cHuman Papillomavirusxe2x80x9d, New York, Oxford Press, pp 9-39; and National Institutes of Health, Consensus Development Conference Statement on Cervical Cancer, Apr. 1-3, 1996).
The precursor to cervical cancer is dysplasia, also known in the art as cervical intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL) (Brinton et al. (1992) xe2x80x9cEpidemiology of cervical Cancer: Overviewxe2x80x9d in xe2x80x9cThe Epidemiology of Cervical Cancer and Human Papillomavirusxe2x80x9d, Lyon, France: International Agency for Research on Cancer; and Tabbara et al. (1992) xe2x80x9cThe Bethesda classification for squamous intraepithelial lesions: histologic, cytologic and viral correlatesxe2x80x9d, Obstet. Gynecol 79: 338-346). While it is not understood how normal cells become transformed, the concept of a continuous spectrum of histopathological change from normal, stratified epithelium through CIN to invasive cancer has been widely accepted for many years (see, for example, Mitchell et al. (1994) xe2x80x9cThe natural history of cervical intraepithelial neoplasia: an argument of intermediate endpoint biomarkersxe2x80x9d, Cancer Epidmiol. Biomark. Prev. 3: 619-626). A large body of epidemiological and molecular biological evidence has been gathered that establishes human papillomavirus (HPV) infection as a causative factor in cervical cancer (Munoz et al. (1992) in xe2x80x9cThe Epidemiology of Human Papillomavirus and Cervical Cancerxe2x80x9d, IRAC publication no. 119, Lyon France: Int. Agency for Research on Cancer, pp 251-261). HPV is found in 85% or more of squamous cell invasive lesions, which represent the most common histologic type seen in cervical carcinoma (Cox et al. (1995) Baillierre""s Clin. Obstet Gynaecol. 91-37). Additional cofactors include, for example, oncogenes activated by point mutations, and chromosomal translocations of deletions (Spandidos et al. (1989) J. Pathol. 157: 1-10).
Cytological examination of Papanicolaou-stained cervical smears (also referred to as Pap smears) currently is the method of choice for detecting cervical cancer. Despite the historical success of this test, concerns have arisen regarding its ability to predict reliably the behavior of same preinvasive lesions (Ostor et al. (1993) Int. J. Gynecol. Pathol. 12: 186-192; and Genest et al. (1993) Human Pathol 24: 730-736). The identification of a cervical cancer-associated tumor marker for reliably detecting early onset of cervical cancer and/or providing early prognostic information will greatly aid the management of cervical cancer.
All eukaryotic cells have a nucleus containing DNA, or chromatin, which is organized by an internal protein scaffolding known as the nuclear matrix (NM). The nuclear matrix was first described in 1974 by Berezney et al. (Berezney et al. (1974) Biochem. Biophys. Res. Commun., 60: 1410-1417). Penman et al. describe a method for selectively extracting insoluble interior nuclear matrix proteins and their associated nucleic acids from cells and determining the particular cell type by analyzing the proteins by two dimensional gel electrophoresis (see for example, U.S. Pat. No. 4,882,268, issued Nov. 21, 1989, and U.S. Pat. No. 4,885,236, issued Dec. 5, 1989, the disclosures of which are incorporated herein by reference).
The nuclear matrix is believed to be involved in a wide variety of nuclear functions fundamental to the control of gene expression. For a general review see, for example, Fey et al. (1991) Crit. Rev. Euk. Gene Express. 1: 127-143. Tissue-specific nuclear matrix proteins have been identified in the rat, mouse and human. Fey et al. (1986) Proc. Natl. Acad. Sci. USA 85: 121-125; Stuurman et al. (1990) J. Biol. Chem. 265: 5460-5465; and Getzenberg et al. (1990) Mol. Endocrinol. 4: 1336-1342. Changes in the presence or absence of specific nuclear matrix proteins have been associated with cellular transformation and differentiation (Bidwell et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3162-3166; Brancolini et al. (1991) Proc. Natl. Acad. Sci. USA 88: 6936-6940; and Greenfield et al. (1991) Proc. Nat. Acad. Sci. USA 88: 11217-11221).
Several recent studies using similar methodology have identified tumor-specific nuclear a matrix proteins in cancers of the prostate (Partin et al. (1993) Cancer Res. 53: 744-746), breast (Khanuja et al. (1993) Cancer Res. 53: 3394-3398), colon cancer (Keesee et al. (1994) Proc. Natl. Acad. Sci. USA 91: 1913-1916), bone (Bidwell et al. (1994) Cancer Res. 54: 28-32), bladder (Getzenberg et al. (1996) Cancer Res. 56: 690-694) and the larynx (Donat et al. (1996) Otolaryngol. Head Neck Surg. 114: 387-393). Molecular characterization of the specific nuclear matrix proteins, however, remains poorly defined, due to the low abundance of these proteins in the cell and their generally insoluble character.
There is, however, a need in the art for specific, reliable markers that are expressed differentially in normal and cancerous cervical tissue and that may be useful in the detecting cervical cancer or in the prediction of its onset. Accordingly, it is an object of this invention to provide cervical cancer-associated molecules which are useful as markers for the early and/or rapid detection of cervical cancers in an individual. It is another object of this invention to provide methods for detecting cervical cancers in an individual. It is another object of the invention to provide methods and compositions for treating cervical cancers in an individual and for monitoring the efficacy of such a treatment in the individual.
The invention provides a variety of methods and compositions for detecting and/or prognosing cervical cancer in a tissue or body fluid sample of an individual. The invention is based, in part, upon the discovery of cervical cancer-associated proteins which are present at detectable levels in cervical cancer cells, but which are not detectable in normal cervical cells, as determined by two-dimensional gel electrophoresis.
In one aspect, the invention provides a method for detecting cervical cancer in a human. The method comprises the step of detecting the presence of a cervical cancer-associated protein in a tissue or body fluid sample of the human thereby to indicate the presence of a cervical cancer or a precursor of a cervical cancer. The cervical cancer-associated protein is characterized as having a molecular weight of from about 44,900 Daltons to about 69,400 Daltons, as determined by standard polyacrylamide gel electrophoresis techniques and an isoelectric point of from about 5.1 to about 6.6 as determined by standard isoelectric focusing techniques. In addition, the cervical cancer-associated protein is further characterized as being a non-chromatin protein which is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis. It is contemplated, however, that the accuracy and/or reliability of the method may be further enhanced by detecting the presence of a plurality of cervical cancer-associated proteins in the preselected tissue or body fluid sample.
As used herein, the term xe2x80x9ccervical cancerxe2x80x9d is understood to mean any cancer or cancerous lesion associated with cervical tissue or cervical cells and, in addition, includes precusors to cervical cancer, for example, dysplasia (also known in the art as a cervical intraepithelial neoplasia or a squamous intraepithelial lesion).
As used herein, the term xe2x80x9ccervical cancer-associatedxe2x80x9d molecules refers to molecules originating from and isolatable from a cervical cancer cell or cells, and substantially neither originating from nor isolatable from a normal cervical cancer cell or cells. As used herein, the term xe2x80x9ccervical cancer-associated proteinxe2x80x9d is understood to mean any protein which is detectable at a higher level in cervical cancer cells than in normal cervical cells, as determined by two-dimensional (2-D) gel electrophoresis. It is not necessary that the target molecule or target protein be unique to a cervical cancer cell; rather it is preferred that the target molecule or protein has a signal to noise ration high enough to discriminate between samples originating from a cervical cancer tissue or body fluid and samples originating from normal cervical tissue or body fluid.
In a preferred embodiment, methods of the invention comprise the step of detecting one or more cervical cancer (CvC) associated proteins, referred to herein as CvC-1 through CvC-5, which can be purified or co-purified using nuclear matrix protein purification methodologies, well known and thoroughly documented in the art. See, for example, Fey et al. (1986) Proc. Natl. Acad. Sci, USA 85: 121-125, the disclosure of which is incorporated herein by reference. As used herein, the term xe2x80x9cnuclear matrix proteinxe2x80x9d is understood to mean any non-cytoskeletal, non-lamin, non-chromatin protein that (i) is isolated from mammalian cell nuclei, (ii) is resistant to solubilization from the nuclei in 0.25M ammonium sulfate, (iii) remains in solution following dialysis into physiological buffer from 8M urea and (iv) is detectable on a silver stained two-dimensional electrophoresis gel. Accordingly, one or more of the resultant cervical cancer-associated proteins may be further defined as being a nuclear matrix protein.
In a preferred embodiment, methods of the invention may comprise the step of detecting the protein CvC-1, a protein having a molecular weight of about 69,400 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.8, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-2, a protein having a molecular weight of about 53,800 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.5, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-3, a protein having a molecular weight of about 47,900 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.6, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-4, a protein having a molecular weight of about 46,000 Daltons, as determined by polyacrylamide gel electrophoresis, and a pl of about 5.1, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-5, a protein having a molecular weight of about 44,900 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 6.6, as determined by isoelectric focusing techniques.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 1; SEQ ID NO.: 2; SEQ ID NO.: 3; SEQ ID NO.: 4; SEQ ID NO.: 5; SEQ ID NO.: 6; SEQ ID NO.: 7; SEQ ID NO.: 8; and SEQ ID NO.: 9. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 10, commonly by referred to in the art as IEF SSP 9502. See, for example, Honore et al. (1994) Gene 151: 291-296, the disclosure of which is incorporated herein by reference.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 11; SEQ ID NO.: 12; SEQ ID NO.: 13; SEQ ID NO.: 14; SEQ ID NO.: 15; SEQ ID NO.: 16; and SEQ ID NO.: 17. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 18, and commonly referred to in the art as Cytokeratin 17. See, for example, Troyanovsky et al. (1992) J. Biol. Biol. 59: 127-137, the disclosure of which is incorporated herein by reference.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 19; SEQ ID NO.: 20; SEQ ID NO.: 21; SEQ ID NO.: 22; SEQ ID NO.: 23; SEQ ID NO.: 24; and SEQ ID NO.: 25. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 26, commonly referred to in the art as TDP-43. See, for example, Ou et al. (1995) J. Virol. 69: 3584-3596, the disclosure of which is incorporated herein by reference.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 27; SEQ ID NO.: 28; SEQ ID NO.: 29; SEQ ID NO.: 30; SEQ ID NO.: 3 1; SEQ ID NO.: 32; and SEQ ID NO.: 33. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 34, commonly referred to in the art as Nup358. See, for example, Wu et al. (1995) J. Biol. Chem. 270: 14209-14213, the disclosure of which is incorporated herein by reference.
In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 35; SEQ ID NO.: 36; SEQ ID NO.: 37; SEQ ID NO.: 38; SEQ ID NO.: 39; SEQ ID NO.: 40; SEQ ID NO.: 41; SEQ ID NO.: 42; SEQ ID NO.: 43; SEQ ID NO.: 44; and SEQ ID NO.: 45. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 46, commonly referred to in the art as lamin A. See, for example, Fisher et al. (1 986) Proc. Natl. Acad. Sci. USA. 83: 6450-6454, the disclosure of which is incorporated herein by reference.
The methods of the invention may be performed on any relevant tissue or body fluid sample. For example, methods of the invention may be performed on cervical tissue, more preferably cervical biopsy tissue, and most preferably on Pap smears. Alternatively, the methods of the invention may be performed on a human body fluid sample selected from the group consisting of: blood; serum; plasma; fecal matter; urine; vaginal secretion; spinal fluid; saliva; ascitic fluid; peritoneal fluid; sputum; and breast exudate. It is contemplated, however, that the methods of the invention also may be useful in assays for metastasized cervical cancer cells in other tissue or body fluid samples.
Marker proteins associated with a cervical cancer in a tissue or body fluid sample may be detected using any of a number of assay methods available in the art. In one embodiment, for example, the marker cervical cancer-associated protein may be reacted with a labeled binding moiety capable of specifically binding to the marker protein thereby to produce a labeled complex of the binding moiety and the marker protein. The labeled complex thereafter may be detected, using conventional methodologies well known in the art. Detection of the presence of the labeled complex may provide an indication of the presence of the cervical cancer cells or pre-cancerous cells in the individual being tested. As used herein, the term xe2x80x9cbinding moietyxe2x80x9d is understood to mean any binding partner capable of specifically binding to a cervical cancer-associated protein with a binding affinity greater than about 105 Mxe2x88x921. As used herein the terms xe2x80x9cspecifically bindingxe2x80x9d, xe2x80x9cspecifically boundxe2x80x9d and xe2x80x9cbinds specificallyxe2x80x9d refer to a binding interaction a with a binding affinity of greater than about 105 Mxe2x88x921. As used herein, the binding moiety is labeled with a detectable moiety, for example, a radioactive, fluoroscopic, spectroscopic, or enzymatic label, using techniques well known in the art.
It is appreciated that, binding moieties which interact and bind specifically with the target protein, may be designed using conventional methods well known in the art. In the invention, the binding moiety can be an antibody, for example, a monoclonal or a polyclonal antibody. Monoclonal antibodies are preferred. It is contemplated, however, that other useful binding moieties useful in the practice of the instant invention may include, for example, biosynthetic antibody binding sites, also referred to in the art as BABS or sfv""s, and antibody fragments, for example, Fv, Fab, Fabxe2x80x2 and (Fabxe2x80x2)2 fragments. Procedures for preparing, testing, and labeling BABS and antibody fragments are well known in the art, and so are not discussed in detail herein.
In another embodiment, one or more marker proteins in a sample may be detected by first isolating the proteins from the sample, and then separating the proteins by two-dimensional gel electrophoresis to produce a characteristic two-dimensional gel electrophoresis pattern. The gel electrophoresis pattern then may be compared with a standard, for example, a standard gel pattern obtained from a data base of gel electrophoresis patterns. Thus, in another embodiment, the invention provides electrophoresis gel patterns or electropherograms of cervical cancer-associated proteins which are useful in detecting a cervical cancer in an individual.
The cervical cancer-associated proteins of the invention can be purified or co-purified from cervical cancer cells using nuclear matrix protein isolation procedures, such as those disclosed in U.S. Pat. Nos. 4,885,236 and 4,882,268, the disclosures of which are incorporated herein. Alternatively, the marker proteins, once identified and characterized may be isolated from the sample by any of a range of protein purification protocols well known to those skilled in the art, such as affinity chromatography, to yield isolated proteins. As used herein, the term xe2x80x9cisolatedxe2x80x9d is understood to mean substantially free of desired, contaminating proteinaceous material.
Furthermore, the skilled artisan may produce nucleic acid sequences encoding the entire isolated marker protein, or fragments thereof, using methods currently available in the art (see, for example, Maniatis et al., eds. (1989) xe2x80x9cMolecular Cloning: A Laboratory Manual,xe2x80x9d Cold Spring Harbor Press). For example, an isolated cervical cancer-associated protein may be sequenced using conventional peptide sequencing protocols, and then oligonucleotide hybridization probes designed for screening a cDNA library. The cDNA library then may be screened with the resultant oligonucleotide to isolate full or partial length cDNA sequences which encode the isolated protein.
Furthermore, the skilled artisan, using the methodologies described in U.S. Pat. Nos. 4,885,236 and 4,882,268 may isolate from a cell sample a nucleic acid molecule having a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein. In such a procedure, the soluble proteins are separated from the nucleus and cytoskeleton by extracting mammalian cells with a non-ionic detergent solution at physiological pH and ionic strength. The insoluble protein and nucleic acids then are digested with DNAase and then eluted with a buffered ammonium sulfate solution to yield a nucleic acid molecule capable of recognizing and being specifically bound by a cervical cancer-associated protein. Any remaining proteins then arc separated from the target nucleic acid molecule.
Detection of the aforementioned nucleic acid molecules thus can serve as an indicator of the presence of cervical cancer and/or metastasized cervical cancer in an individual. Accordingly, in another aspect, the invention provides another method for detecting cervical cancer in a human. The method comprises the step of detecting the presence of a nucleic acid molecule in a tissue or body fluid sample thereby to indicate the presence of a cervical carcinoma in the individual. The nucleic acid molecule is selected from the group consisting of (i) a nucleic acid molecule comprising a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein, and (ii) a nucleic acid molecule comprising a sequence encoding a cervical cancer-associated protein. As defined herein, the cervical cancer-associated protein is characterized as being selected from the group consisting of (i) a protein having a molecular weight of about 69,400 Daltons and an isoelectric point of about 5.8; (ii) a protein having a molecular weight of about 53,800 Daltons and an isoelectiic point of about 5.5; (iii) a protein having a molecular weight of about 47,900 Daltons and an a isoelectric point of about 5.6; (iv) a protein having a molecular weight of about 46,000 Daltons, and an isoelectric point of about 5. 1; and (v) a protein having a molecular weight of about 44,900 Daltons and an isoelectric point of about 6.6, wherein in each example, the molecular weight is determined by standard polyacrylamide gel electrophoresis techniques and the isoelectric point is determined by standard isoelectric focusing techniques, and wherein the cervical cancer-associated protein is further characterized as being a non-chromatin protein which is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis.
A target nucleic acid molecule in a sample may be detected, for example, by Northern blot analysis by reacting the sample with a labeled hybridization probe, for example, a 32P labeled oligonucleotide probe, capable of hybridizing specifically with at least a portion of the nucleic acid molecule encoding the marker protein. Detection of a nucleic acid molecule either encoding a cervical cancer-associated protein or capable of being specifically bound by a cervical cancer-associated protein, thus can serve as an indicator of the presence of a cervical cancer in the individual being tested.
In another aspect, the invention provides a kit for detecting the presence of cervical cancer or for evaluating the efficacy of a therapeutic treatment of a cervical cancer. Such kits may comprise, in combination, (i) a receptacle for receiving a human tissue or body fluid sample from the individual, (ii) a binding partner which binds specifically either to an epitope on a marker cervical cancer-associated protein or a nucleic acid sequence encoding at least a portion of the marker cervical cancer-associated protein, (iii) means for detecting the binding of the binding partner with either the cervical cancer-associated protein or the nucleic acid sequence encoding at least a portion of the cervical cancer-associated protein, and (iv) a reference sample.
In one embodiment of the kit, the binding moiety binds specifically to a cervical cancer-associated protein selected from the group of proteins further defined as having: a molecular weight of about 69,400 Daltons and an isoelectric point of about 5.8; a molecular weight of about 53,800 Daltons and an isoelectric point of about 5.5; a molecular weight of about 47,900 Daltons and an isoelectric point of about 5.6; a molecular weight of about 46,000 Daltons and an isoelectric point of about 5.1, or a molecular weight of about 44,900 Daltons and an isoelectric point of about 6.6, wherein the molecular weight is determined by conventional polyacrylamide gel electrophoresis methodologies, and the isoelectric point is determined by conventional isoelectric focusing methodologies.
In another embodiment of the kit, the reference sample may comprise a negative and/or positive control. The negative control being indicative of a normal cervical cell type and the positive control being indicative of cervical cancer.
In another aspect, the invention provides a method for treating cervical cancer. The method comprises administering to a patient with cervical cancer, a therapeutically-effective amount of a compound, preferably an antibody, and most preferably a monoclonal antibody, which binds specifically to a target cervical cancer-associated protein thereby to inactivate the protein. The target protein being characterized as having a molecular weight of from about 44,900 Daltons to about 69,400 Daltons, as determined by standard polyacrylamide gel electrophoresis techniques and an isoelectric point of from about 5.1 to about 6.6, as determined by standard isoelectric focusing techniques, and wherein the target protein is further characterized as being a non-chromatin protein which is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis. Similarly, it is contemplated that the compound may comprise a small molecule, for example, as small as organic molecule, which inhibits or reduces the biological activity of the target cervical cancer-associated protein.
In another aspect, the invention provides another method for treating cervical cancer. The method comprises the step of administering to a patient diagnosed as having cervical cancer, a therapeutically-effective amount of a compound which reduces in vivo the expression of a target cervical cancer-associated protein thereby to reduce in vivo the expression of the target protein. In a preferred embodiment, the compound is a nucleobase containing sequence, such as, an anti-sense nucleic acid sequence or anti-sense peptide nucleic acid (PNA) molecule, complementary to a nucleic acid sequence encoding at least a portion of the target protein. After administration, the anti-sense nucleic acid sequence or anti-sense PNA molecule binds to the nucleic acid sequences encoding, at least in part, the target protein thereby to reduce in vivo expression of the target cervical cancer-associated protein.
Thus, the invention provides a wide range of methods and compositions for detecting and treating cervical cancer in an individual. Specifically, the invention provides cervical cancer-associated proteins, which permit specific and early, preferably before metastases occur, detection of cervical cancer in an individual. In addition, the invention provides kits useful in the detection of cervical cancer in an individual. In addition, the invention provides methods utilizing the cervical cancer-associated proteins as targets and indicators, for treating cervical cancers and for monitoring of the efficacy of such a treatment. These and other numerous additional aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims which follow.